TPL-2 Inhibits IFN-β Expression via an ERK1/2-TCF-FOS Axis in TLR4-Stimulated Macrophages

Key Points TPL-2 activation of ERK1/2 regulates gene expression in TLR-stimulated macrophages. TPL-2 regulates transcription via ERK1/2 phosphorylation of ternary complex factors. TPL-2 inhibits Ifnb1 transcription via ternary complex factor–induced Fos mRNA expression.

M acrophages are a critical cellular component of the immune system, responding rapidly to pathogens to initiate innate immune responses (1). Following infection, macrophages are stimulated by invariant pathogen molecules, termed pathogen-associated molecular patterns, via pattern recognition receptors. For example, LPS from Gram-negative bacterial cell walls binds to the pattern recognition receptor TLR4, expressed at the plasma membrane and on endosomes in macrophages (2). TLR stimulation of macrophages triggers the coordinate activation of several intracellular signaling pathways and the expression of multiple genes, which include the cytokines and chemokines responsible for promoting an inflammatory response and coordinating an effective adaptive immune response (3).
TLR stimulation results in the activation of the IkB kinase (IKK) complex, leading to the nuclear translocation of NF-kB transcription factor dimers, and the activation of classical MAPKs ERKs 1 and 2 (ERK1/2), JNKs 1 and 2 (JNK1/2), and p38a (4,5). NF-kB and MAPK activation together promote expression of multiple proinflammatory cytokines. Stimulation of certain TLRs, including TLR3 and TLR4, also activates TANK binding kinase 1 (TBK1), which phosphorylates IFN regulatory factors (IRFs) and drives the expression of type I IFNs.

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TPL-2 TRANSCRIPTOME TLR4 stimulation, the p38a-activating kinase MKK4 is activated independently of TPL-2. Consequently, TLR4 activation of p38a in macrophages is only marginally reduced by the absence of TPL-2 signaling, whereas ERK1/2 activation is blocked. Analyses of Tpl2 −/− macrophages indicate that TPL-2 stimulates the expression of a number of inflammatory mediators following LPS stimulation of macrophages, including IL-1b, TNF, CCL2, CXCL2, and COX2 (1315). Pharmacological inhibition of MKK1/2 inhibition phenocopies the effects of TPL-2 deficiency, suggesting that TPL-2 controls TLR4 induction of these genes via ERK1/2 activation. Consistent with these inhibitory effects on proinflammatory gene expression, TPL-2 deficiency reduces inflammation in a number of preclinical disease models. Tpl2 −/− mice are protected from LPS-induced septic shock, TNF-induced inflammatory bowel disease, experimental autoimmune encephalomyelitis, acute pancreatitis, and liver injury (1418). TPL-2 is consequently considered a potential anti-inflammatory drug target, particularly for inflammatory bowel disease, the development of which is linked genetically to MAP3K8 (19).
TPL-2 signaling also has inhibitory effects on gene expression. For example, we have previously shown that TPL-2 activation of ERK1/2 inhibits IFN-b transcription in LPS-stimulated macrophages and DCs (20). Subsequent experiments underlined the importance of this negative regulation, demonstrating that TPL-2 inhibition of type I IFN signaling is essential for effective innate immune responses to Mycobacteria tuberculosis and Listeria monocytogenes (21). Consistent with this, type I IFNs are known to have a detrimental effect on immune responses to these bacteria, abrogating IFN-gdependent host-protective immune responses via downregulation of the IFN-g receptor, while promoting the production of the anti-inflammatory cytokine IL-10 (22).
The mechanisms by which TPL-2 signaling alters transcription is not known. In this study, we used RNA sequencing (RNA-seq) to demonstrate that ternary complex factors (TCFs) have an important role in mediating the effects of the TPL-2 MAPK pathway on gene expression in TLR4-stimulated macrophages. TCFs are direct targets of the RAFERK1/2 MAP kinase pathway regulating immediate early gene transcription and a gene expression program that controls proliferation in serum-stimulated fibroblasts (23,24). The results of this study demonstrate that TCFs also control gene expression in TLRstimulated primary mouse macrophages downstream of the TPL-2ERK1/2 MAPK pathway, suggesting an important regulatory role for this family of transcription factors in innate immune responses.

Mouse strains
Mouse strains were bred in a specific-pathogen-free environment at the Francis Crick Institute (London, U.K.), and all experiments were done in accordance with regulations of the U.K. Home Office. Nfkb1 SSAA/SSAA , Fos fl/fl , Elk1 −/− Elk4 −/− , LysM-Cre, and Map3k8 D270A/D270A mouse strains have been described previously (11,2527) and were all fully backcrossed onto a C57BL/6 background.

RNA-seq
RNA was isolated using the RNeasy kit (QIAGEN). Biological replicate libraries were prepared using the TruSeq RNA library prep kit (Illumina) and were single-end sequenced on the Illumina HiSeq 2500 platform. Sequencing yield was typically ∼25 million strand-specific reads. The RSEM package (version 1.2.31) (28) in conjunction with the STAR alignment algorithm (version 2.5.2a) (29) was used for the mapping and subsequent gene-level counting of the sequenced reads with respect to mm10 RefSeq genes downloaded from the UCSC Table Browser  Quantitative RT-PCR RNA was isolated using the RNeasy kit (Qiagen). Five hundred to 1000 ng of RNA was reverse transcribed using the SuperScript VILO cDNA synthesis kit (Invitrogen). mRNA expression was measured using TaqMan probes and TaqMan gene expression master mix (Applied Biosystems) on Quant-Studio 3 and 5 real-time PCR systems (Applied Biosystems). Data were normalized to hypoxanthine phosphoribosyltransferase using the cycle threshold (DDCt) method.

IFN-b ELISA
Secreted IFN-b in tissue culture supernatant was quantified using the Veri-Kine mouse IFN-b ELISA kit (PBL Assay Science) as per the manufacturer's instructions.

In vivo challenge with LPS
Fos knockout (FosKO) mice and LysM-Cre controls, aged between 10 and 14 wk, were injected i.p. with 5 mg/kg LPS (Escherichia coli O111:B4, Sigma-Aldrich). After 4 h, mice were sacrificed via cervical dislocation and blood was extracted from the heart using a 25G needle and 0.3-ml syringe. Blood was left to coagulate for 30 min and then centrifuged at 17,000 × g for 1 min. Serum was stored at −80 • C until the assay for IFN-b by ELISA.

Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) assays were performed as described previously (23). Briefly, 2-× 15-cm plates of 14 × 10 6 cells were crosslinked. Samples were sonicated using 10 cycles of 30 s on, 30 s off. Samples were precleared for 1 h prior to overnight incubation with Abs. Samples were washed and then crosslinks were reversed overnight. Samples were then purified using a PCR purification kit (QIAGEN) before analysis using quantitative PCR. Percentage input was calculated and then data were normalized and pooled from three or four experiments. The following Abs were used: Rbp1 NTD (D8L4Y) (Cell Signaling Technology), ELK1 (E277) (Abcam), and SRF (1681-1-AP, Lot 1) (Proteintech).

TransAM ELISA assay
For ELISA of NF-kB and AP-1 activity, nuclear extracts were prepared using a commercial kit (Active Motif). Five micrograms of extract per point was assayed in duplicate, using a TransAm NF-kB or AP-1 kit (Active Motif), monitoring RelA and FOS binding, respectively.

Immunoblotting
Immunoblotting of cell lysates was carried out as described previously (33).

Statistical analysis
Analyses of quantitative PCR and ELISA data were performed using Graph-Pad software (GraphPad Software, San Diego, CA). Data were compared using one-way or two-way ANOVA as appropriate. A p value <0.05 or <0.001 was considered significant (*p < 0.05, **p < 0.01). Error bars represent SEM.

Regulation of gene expression in LPS-stimulated macrophages by TPL-2 catalytic activity
Wild-type (WT) BMDMs and Map3k8 D270A/D270A BMDMs, which express kinase-inactive TPL-2 D270A (16) Table I). The number of genes positively and negatively regulated by TPL-2 catalytic activity increased as the time of LPS stimulation increased (Fig. 1A).
To gain some insight into the potential impact of TPL-2 signaling on the biological response of macrophages to LPS stimulation, the genes regulated by TPL-2 catalytic activity were analyzed for process and pathway enrichment using the MetaCore program (Supplemental Fig.  1B). Immune response pathways, in particular cytokine and growth factor signaling via MAPK, NF-kB, and JAK-STAT signaling pathways, and damage-/injury-associated pathways were highly enriched. Similarly, immune response, inflammation, and signaling processes were enriched. These results are consistent with the known importance of TPL-2 in innate immunity, adaptive immunity, and autoimmune inflammation, based on experiments with Map3k8 −/− mice (7).
TPL-2regulated genes were grouped into 12 distinct kinetic groups by k-means cluster analysis (Fig. 1B). Because TPL-2 signaling is rapidly and transiently induced following LPS stimulation of macrophages (33), we focused on clusters 1, 3, and 4, which contained genes that were clearly induced at the early time points following LPS stimulation (Supplemental Fig. 2). Cluster 1 contained the genes most rapidly induced by LPS stimulation and these were strongly reduced by Map3k8 D270A mutation. RNA-seq determined that LPS induction of cluster 1 genes was also significantly reduced by Nfkb1 SSAA mutation (11), which prevents signal-induced proteolysis of p105 and consequently prevents TPL-2 activation of MKK1/ 2 and their substrates ERK1/2 (Fig. 1C). Similarly, pharmacological inhibition of ERK1/2 activation in WT cells with PD0325901 MKK1/2 inhibitor reduced the expression of cluster 1 genes (Fig.  1C). Pharmacological inhibition of RSK1/2 with RSKi47 (34) did not alter LPS-induced expression of mRNAs encoding cluster 1 genes (Fig. 1C), indicating that TPL-2 did not induce cluster 1 gene expression as a consequence of ERK1/2-mediated activation of RSK1/2. Genes in clusters 3 and 4 had a delayed induction compared with cluster 1, but their expression was similarly reduced by Map3k8 D270A and Nfkb1 SSAA mutation, as well as by PD0325901 treatment. Inhibition of RSK1/2 activity with RSKi47 also reduced expression of a fraction of cluster 3 and 4 genes. Interestingly, suppressed expression of cluster 11 genes by TPL-2 catalytic activity was not phenocopied by MKK1/2 inhibition, suggesting that TPL-2 regulated the constitutive expression of a subset of genes independently of ERK1/2 activation.
The overlaps between the effects of Map3k8 D270A mutation and Nfkb1 SSAA mutation or PD0325091 treatment on the expression of individual genes in clusters 1, 3, and 4 were compared quantitatively. At 1 h of LPS stimulation, the expression values in cluster 1 correlated well between Map3k8 D270A/D270A and Nfkb1 SSAA/SSAA cells (r 5 0.748) and between Map3k8 D270A/D270A cells and WT cells 1 PD0325901 (r 5 0.897) (Fig. 1D). Similar comparisons in the expression values for clusters 3 and 4 were also well correlated (Fig. 1D). The RNA-seq experiments therefore supported the hypothesis that TPL-2 catalytic activity promoted the expression of rapidly induced genes in clusters 1, 3, and 4 via activation of ERK1/2, which was dependent on IKK-induced proteolysis of NF-kB1 p105, an essential step for TPL-2 activation of ERK1/2 following LPS stimulation (11). The expression values in clusters 1 and 3 only weakly or moderately correlated between Map3k8 D270A/D270A cells and WT cells 1 RSKi47, suggesting that RSKs did not play a major role in regulating the expression of these gene clusters. However, the effects of Map3k8 D270A mutation and RSKi47 correlated more strongly for cluster 4 genes, suggesting that TPL-2 activation of RSKs contributed to the expression of these genes.

TPL-2 catalytic activity induces the expression of TCF target genes
Studies in fibroblasts have demonstrated that ERK1/2 directly phosphorylates ∼50 transcription factors following growth factor receptor stimulation, including ATF2, C/EBPb, ELK1, ERF, c-FOS, HIF1a, SP1, and c-JUN (35). The mechanisms by which TPL-2 activation of ERK1/2 regulates transcription in macrophages are not understood. We used a bioinformatics approach to investigate this question, focusing on TPL-2regulated genes in cluster 1 that were most rapidly induced by LPS stimulation and therefore likely to be direct targets of the TPL-2ERK1/2 signaling pathway. Analysis of these genes using the MetaCore database revealed that ELK3, ELK4, and  Cluster: ELK1 were among the four most enriched transcription factors (based on a ratio of observed/expected hits) ( Fig. 2A). ELK3, ELK4, and ELK1 belong to the TCF subfamily of ETS family transcription factors (36). Experiments in fibroblasts have demonstrated that TCFs bind cooperatively with serum response factor (SRF) to serum response elements in the promoters of immediate early genes (37). Mitogenic stimulation activates the Ras-Raf MAPK pathway, inducing ERK1/ 2 phosphorylation of C-terminal transactivation domains of TCFs, which triggers their transcriptional activation and immediate early gene expression (38). Immunoblotting of macrophage cell lysates demonstrated that LPS stimulation induced ELK1 phosphorylation at Ser 390 , an ERK1/2 phosphorylation site, and this was blocked by Map3k8 D270A mutation (Fig. 2B). LPS-induced ELK1 Ser 390 phosphorylation was also prevented by PD0325901 MKK1/2 inhibitor treatment of WT cells and by Nfkb1 SSAA mutation (Fig. 2B, 2C). These data indicated that TPL-2 signaling induced C-terminal ELK1 phosphorylation by ERK1/2, downstream of IKK-induced NF-kB1 p105 proteolysis.
Analysis of the RNA-seq data revealed that Map3k8 D270A mutation significantly reduced the mRNA abundance of the TCF-regulated immediate early genes Egr1, Egr3, and Nr4A1 following LPS stimulation of macrophages (Fig. 2D), which was confirmed by quantitative RT-PCR (qRT-PCR) (Fig. 2E). Nfkb1 SSAA mutation and treatment of WT cells with PD0325901 also reduced the LPS-induced expression of Egr1, Egr3, and Nr4A1 mRNAs in macrophages (Fig. 2E, 2F). Primary transcript qRT-PCR demonstrated that Map3k8 D270A mutation reduced mRNA levels of these genes by impairing their transcription (Supplemental Fig. 3A). Consistent with this, ChIP experiments showed that LPS stimulation induced recruitment of RNA polymerase II to the promoters of Egr1, Egr3, and Nr4A1, which was reduced by Map3k8 D270A mutation (Fig. 2G). Binding of ELK1 to the Egr1, Egr3, and Nr4a1 promoters was fractionally increased in LPS-stimulated WT cells, and this increase was blocked by Tpl2 D270A mutation (Fig. 2H). In contrast, binding of SRF was constitutive to the Egr1, Egr3, and Nr4a1 promoters and unaffected by Map3k8 D270A mutation (Fig. 2I). Taken together, these results indicated that TLR4 activation of the IKK2NF-kB1 p105TPL-2ERK1/2 MAPK pathway promoted TCF phosphorylation, thereby inducing transcription of TCF/ SRF-regulated immediate-early genes.

ELK1/ELK4 regulate nearly half of TPL-2dependent genes in LPSstimulated macrophages
The roles of TCFs in the regulation of gene expression have been intensively studied in serum-stimulated fibroblasts (23,39). However, the contribution of TCFs to gene expression in innate immune responses is not known. ELK1 and ELK4 act redundantly and their knockout phenotypes are distinct from those of ELK3 (27,40). We therefore analyzed TLR4-induced gene expression in macrophages generated from Elk1 −/− Elk4 −/− mice. As expected, qRT-PCR demonstrated that LPS-induced expression of Egr1, Egr3, and Nr4a1 mRNAs was significantly reduced in Elk1 −/− Elk4 −/− macrophages compared with WT cells (Fig. 3A).
In total, 211 genes were found to be >2-fold coregulated by TPL-2 and TCFs. Analysis of these genes using the MetaCore program revealed an enrichment for processes involving Th17 cytokines, chemotaxis, signaling, and proliferation (Supplemental Fig. 3B). The pathway enrichment analysis demonstrated the involvement of these genes in a range of immune and inflammation pathways, including IL-1 signaling and inflammatory responses associated with asthmatic airway fibroblasts. This analysis suggests that TCFs activated by the TPL-2ERK1/2 MAPK pathway in macrophages can regulate genes that control a number of aspects of innate immune responses.

Regulation of Fos family genes by the TPL-2ERK1/2TCF signaling pathway
Experiments with TCF-deficient fibroblasts have shown that >60% of 12-O-tetradecanoylphorbol-13-acetate (TPA)induced gene transcription is mediated via ERK1/2 activation of TCFs (23). Direct TCF-SRF targets in these cells encompass 54 different transcription factors, suggesting that TCFs control transcription of many genes indirectly via induced expression of these downstream transcription factors, which include members of the Egr, AP-1, Ets, and NF-kB families.
MetaCore analysis of TPL-2regulated genes in gene clusters 1, 3, and 4 in LPS-stimulated macrophages suggested a role for AP-1 transcription factors in mediating the transcriptional output of TLP-2 ( Fig. 2A, Supplemental Fig. 4A, 4B). The AP-1 family is composed of 15 polypeptides, which include the FOS, JUN, and ATF subfamilies (41), which bind to DNA as obligate dimers via conserved bZIP domains. Binding sites for all FOS family members (FOS, FRA-1, FRA-2, and FOS-B) were enriched across the promoters of TPL-2regulated genes in clusters 1 and 4. In addition, ATF2 and ATF3 binding sites were enriched in clusters 4 and 3 gene promoters, respectively, and JUN, JUN-B, and JUN-D binding sites were enriched in the promoters of cluster 4 genes.
Analysis of the Map3k8 D270A/D270A /WT RNA-seq dataset indicated that TPL-2 signaling promoted the LPS-induced mRNA expression of all Fos and Jun family members (Supplemental Fig. 4C). Map3k8 D270A mutation significantly reduced Fos and FosB mRNA levels at 0.5 and 1 h of LPS stimulation, whereas Fra1 (Fosl1) and  gene mRNAs following LPS stimulation of macrophages in a similar fashion to Map3k8 D270A mutation (Fig. 4A, Supplemental  Figs. 4C, 5A). The pronounced effects on the expression of Fos genes suggested that TCF control of gene expression in LPS-stimulated macrophages was mediated via control of Fos family transcription. Because Fos is a known direct target of TCF-mediated transcription (42) and Fos mRNA was expressed at much higher levels in LPS-stimulated macrophages than FosB, Fra1, and Fra2 (Supplemental Fig. 4C), we focused on the role of FOS in mediating TPL-2 regulation of gene expression.
To determine the contribution of FOS to LPS-induced gene expression, BMDMs generated from Fos FL/FL LysM-Cre (FosKO) and Fos FL/1 LysM-Cre (LysM-Cre) control mice were stimulated with LPS and gene expression was analyzed by RNA-seq. FOS deficiency significantly reduced LPS-induced Fra1 and Fra2 mRNA levels while delaying LPS induction of FosB mRNA (Supplemental Fig. 5B). The absence of FOS, therefore, significantly reduced expression of other Fos family genes, as previously shown (43,44).
To determine whether TPL-2 regulates gene expression via a TCF-FOS signaling axis in LPS-stimulated macrophages, genes modulated by Tpl2 D270A mutation were compared with genes modulated by TCF deficiency and FOS deficiency (Fig. 4G). Twenty-nine percent (44/153) of the genes that were downregulated both in Tpl2 D270A/D270A and Elk1/4 −/− cells relative to WT cells were also downregulated in FosKO BMDMs compared with LysM-Cre controls. In addition, >50% (30/58) of genes upregulated in both Tpl2 D270A/D270A and Elk1/4 −/− BMDM RNA-seq datasets were also upregulated in FOS-deficient cells. These results indicate that a significant fraction of genes regulated by TPL-2 catalytic activity in LPS-stimulated macrophages were controlled via FOS by TCFmediated Fos transcription. LPS stimulation induced phosphorylation of FOS on two ERK1/2-regulated sites (Ser 362 /Ser 374 ) (data not shown), suggesting that TPL-2 may have also regulated FOS levels via ERK1/2 regulation of FOS protein stability (45).

Inhibition of type I IFN expression by the TPL-2TCFFOS signaling pathway
To calculate enrichment of FOS-regulated genes within particular pathways, FOS-regulated genes at 0.5 h of LPS stimulation were analyzed using the MetaCore program. The three most significantly enriched pathways among genes upregulated by FOS deficiency at 0.5 h were all linked to type I IFN signaling (Supplemental Fig.  5C). Gene set enrichment analysis also identified type I IFN signaling as the most enriched pathway among TPL-2regulated cluster 4 genes (data not shown), in which several FOS family members were identified as potential transcription factors.
Analysis of the FosKO/LysM-Cre RNA-seq dataset revealed that FOS deficiency significantly augmented LPS induction of Ifnb1 mRNA (Fig. 5A). qRT-PCR confirmed that FOS deficiency augmented Ifnb1 mRNA expression after LPS stimulation of BMDMs (Fig. 5B), with increased IFN-b protein detected in culture supernatants (Fig. 5C). Serum levels of IFN-b protein were also significantly increased in FosKO mice compared LysM-Cre controls after LPS injection, confirming the physiological relevance of the in vitro findings. mRNA levels of multiple IFN-stimulated genes (ISGs) (46) were elevated at 2 h of LPS in FosKO BMDMs compared with control LysM-Cre control cells (Supplemental Fig. 5D). Furthermore, mRNA levels of Ifna2, Ifna4, Ifna5, Ifna6, and Infa13, which were not induced by LPS in control cells, were increased following LPS stimulation of FosKO BMDMs (Supplemental Fig. 6). These results indicated that FOS deficiency induced a type I IFN signature in LPS-stimulated macrophages by promoting the expression of type I IFNs. The expression of a subset of ISGs was increased by FOS deficiency in unstimulated cells (Supplemental Fig. 5D), possibly suggesting direct suppression of specific ISG expression by FOS in the absence of type I IFN production.
Consistent with our earlier observations with Tpl2 −/− BMDMs (20), analysis of Map3k8 D270A /WT RNA-seq data demonstrated that Map3k8 D270A mutation increased Ifnb1 mRNA levels in BMDMs following LPS stimulation (Fig. 5E). qRT-PCR quantitation confirmed this finding and also showed that LPS-induced expression of Ifnb1 mRNA was increased by PD0325901 treatment of WT BMDMs and by Nfkb1 SSAA mutation (Fig. 5F, 5G). Furthermore, TCF deficiency (Elk1/4 −/− /WT BMDM RNA-seq comparison) significantly increased Ifnb1 mRNA after LPS stimulation (Fig. 5E). Taken together, these results show that TPL-2 repression of Ifnb1  To determine the broader significance of TPL-2 and FOS in regulating Ifnb1 mRNA expression in innate immune responses, BMDMs were stimulated with CpG (TLR9), Pam 3 CSK 4 (TLR1/2), or TNF. Map3k8 D270A mutation reduced Fos mRNA expression induced by each of these ligands (data not shown) and increased both Ifnb1 mRNA and IFN-b levels (Fig. 5H). Ifnb1 mRNA expression and IFN-b protein secretion were also significantly increased by FOS deficiency following stimulation of BMDMs with each of the ligands (Fig. 5I). These results indicate that the TPL-2 stimulation of FOS expression plays a general role in suppressing type I IFN signaling in innate immune responses of macrophages.

TPL-2 regulates IFN-b transcription via multiple pathways
IFN-b transcription is regulated by the cooperative binding of ATF2-JUN AP-1, IRF3/IRF7, and NF-kB to the Ifnb1 enhanceosome (47). An NF-kB ELISA demonstrated that Map3k8 D270A mutation inhibited Ifnb1 transcription independently of effects on NF-kB activation following LPS stimulation of BMDMs (Fig.  6A). LPS-induced FOS/AP-1 binding was reduced by Map3k8 D270A mutation, as expected (Fig. 6B), while analysis of published ChIP/sequencing data (46) showed that FOS is recruited to the Ifnb1 gene following LPS stimulation of BMDMs (Fig. 6C). This suggested that FOS-containing AP-1 dimers may directly inhibit Ifnb1 transcription.
The TPL-2ERK1/2 signaling pathway (49) and FOS (50) suppress LPS induction of the anti-inflammatory cytokine IL-10. IL-10 has profound inhibitory effects on cytokine production by TLRstimulated macrophages (51). We have previously shown that increased IFN-b production by Mapk3k8 −/− BMDMs results in part from reduced IL-10 production (20). Map3k8 D270A mutation (Fig. 6F) and FOS deficiency (Fig. 6G) also significantly decreased expression of Il10 mRNA induced by LPS stimulation of BMDMs. Reduced IL-10 likely contributed to the increased IFN-b production caused by Map3k8 D270A mutation and FOS deficiency.
Collectively, the results presented in this section suggest that the mechanism by which TPL-2 suppresses IFN-b production by TLR4-stimulated macrophages involves multiple interconnected pathways, which likely converge to ensure a robust control of cytokine production in response to pathogens.

Discussion
In this study, RNA-seq was used to comprehensively identify genes regulated by TPL-2 catalytic activity in LPS-stimulated macrophages. Bioinformatics analysis of these genes using the Meta-Core pathway analysis tool confirmed the importance of TPL-2 in regulating immune responses and inflammation. Our previous analyses of the TPL-2 phosphoproteome demonstrated that ∼85% of protein phosphorylations regulated by TPL-2 catalytic activity are dependent on ERK1/2 activation (10). Consistent with this, RNAseq analyses in this study revealed a strong correlation between the effect of an MKK1/2 inhibitor on WT cells and Map3k8 D270A mutation, indicating that most TPL-2regulated changes in transcription in response to TLR4 activation were mediated via ERK1/ 2 activation. However, analysis of gene expression in WT cells treated with the specific RSK inhibitor RSKi47 did not suggest that ERK1/2 activation of RSK had a dominant role in mediating gene expression changes downstream of TPL-2, but rather that ERK1/2 activation itself was important.
Previous studies have focused on regulation of cytokine and chemokine expression by TPL-2 (13,5254). Notably, our work demonstrated that TPL-2 signaling also directly controls the expression of several key transcription factors, including EGR1, EGR3, NR4A1, IER3, and FOS, which is likely to have wide-ranging effects on macrophage function. For example, IER3 is known to play a role in inflammatory bowel disease and following Leishmania infection (55,56), whereas NR4A1 modulates apoptosis of monocytes (57). TPL-2 also controlled the expression of genes involved in negative feedback regulation of MAPK signaling (e.g., Dusp1, Dusp2, and Dusp5), which have key roles in controlling inflammation (58,59). In addition, TPL-2 regulated enzymes controlling extracellular matrix remodeling and PG synthesis, which are important in immune responses (60,61).
Bioinformatics analysis of cluster 1 genes that contained the genes most rapidly regulated by TPL-2 catalytic activity suggested that the TCF family of transcription factors were involved in controlling their transcription. Consistent with this, phosphorylation of ELK1 on a known ERK1/2 target site was inhibited by Map3k8 D270A mutation (62). There was also a strong correlation between genes dependent on TPL-2 catalytic activity and those dependent on ELK1/4 expression. Around 50% of the genes downregulated by Map3k8 D270A mutation between 0.5 and 2 h after LPS stimulation were similarly reduced by deficiency of ELK1 and ELK4 following LPS stimulation of macrophages. Furthermore, of the genes that were upregulated in Map3k8 D270A/D270A macrophages, ∼40% were similarly upregulated in Elk1 −/− Elk4 −/− macrophages. This demonstrated that TCFs mediate a substantial fraction of TPL-2dependent transcriptional output, either directly or via TCF-dependent transcription of downstream transcription factors. MetaCore analysis of cluster 3 genes identified LITAF (LPSinduced TNF-a factor) (63) as the most enriched transcription factor regulated by TPL-2 catalytic activity. Published work has shown that LITAF forms a complex with STAT6 (64) and is required for LPS induction of cytokines, including TNF-a and IL-6, in macrophages (65). LITAF nuclear translocation is regulated by p38a (65), and the current study raises the possibility that LITAF nuclear translocation and transcription may also be regulated by TPL-2induced activation of ERK1/2. This will be an important area of research for the future to determine how ERK1/2 signaling controls innate immune responses.
Bioinformatics analysis of rapidly induced TPL-2regulated genes in LPS-stimulated macrophages suggested an important role for AP-1 transcription factors in mediating the transcriptional output of TPL-2. AP-1 factors cooperate with other DNA binding proteins to promote transcription, and a recent ChIP-seq analysis indicated that adjacent NF-kB binding motifs are highly correlated with binding of all AP-1 monomers in TLR4-stimulated macrophages (66). Furthermore, there was a statistically significant enrichment of NF-kB target genes, identified by RelA binding within 2 kbp of the transcriptional start site (67), among those genes regulated by TPL-2 catalytic activity in LPS-stimulated BMDMs (data not shown). The coordinated activation of AP-1 with the activation of NF-kB transcription factors via IKK-mediated control of TPL-2 signaling (7) may ensure efficient expression of AP-1/NF-kB target genes in TLR-stimulated macrophages.
Similar to Map3k8 D270A/D270A macrophages, ELK1/ELK4-deficient macrophages have elevated levels of Ifnb1 mRNA compared with WT macrophages following TLR4 stimulation. We have previously shown that FOS overexpression reduces TLR induction of Ifnb1 mRNA in Map3k8 −/− DCs, suggesting an inhibitory role for FOS in Ifnb1 transcription (20). Consistent with this, RNA-seq revealed that Ifnb1 mRNA levels were significantly elevated in LPS-stimulated FOS-deficient macrophages compared with controls, and type I IFN signature pathways were significantly enriched among genes whose expression was increased by the absence of FOS. Furthermore, FOS deficiency facilitated LPS upregulation of IFN-a isoforms in macrophages (which was not evident in LPS-stimulated WT macrophages) and the expression of multiple ISGs was augmented. Taken together, these results indicate that TPL-2 negatively regulates expression of type I IFN, in part by TCF-induced Fos transcription. TPL-2 may additionally control FOS protein stability via ERK1/2mediated FOS phosphorylation, similar to fibroblasts (45).
Ifnb1 transcription involves the coordinated binding of ATF2-JUN AP-1 dimers, IRF3, and NF-kB to the Ifnb1 enhanceosome (47). Map3k8 D270A mutation did not affect LPS activation of NF-kB, although FOS binding to AP-1 was significantly reduced. Analysis of published ChIP sequencing data (46) revealed that LPS stimulation of macrophages induces the recruitment of FOS to the Ifnb1 gene, suggesting that TPL-2 induction of FOS may directly inhibit Ifnb1 transcription. We have previously shown that PPARg transcription can be positively or negatively regulated by AP-1, depending on the composition of the AP-1 dimer (68). The present study suggests that TPL-2 signaling reduces Ifnb1 transcription by controlling the abundance of FOS, which can form inhibitory AP-1 dimers that compete with activating ATF2-containing AP-1 dimers. TPL-2 signaling additionally inhibits IRF3 phosphorylation by TBK1, which will directly reduce Ifnb1 transcription. TPL-2 induction of IL-10 expression via FOS also contributes to the suppression of type I IFN expression by TPL-2 (20,50). TPL-2 signaling, therefore, appears to inhibit type I IFN expression via multiple complementary mechanisms.
FOS-dependent repression of Ifnb1 expression in macrophages was not restricted to LPS stimulation. Other TLR agonists (CpG and Pam 3 CSK 4 ) and TNF suppressed Ifnb1 mRNA levels in macrophages via TPL-2induced FOS expression. Furthermore, LPS-and TNFstimulated increases in Ifnb1 mRNA in fibroblasts derived from mice lacking Elk1, Elk3, and Elk4 were increased relative to WT controls (data not shown). The repression of Ifnb1 transcription by TPL-2 regulation of FOS levels therefore may be of general importance in innate immune responses in myeloid and stromal cells.
It is well established that TCFs regulate proliferation of fibroblasts following growth factor stimulation (23,24) and also control T cell development and function (27). However, a role for TCFs in innate immunity has not been established. The present study identified TCFs and their downstream target FOS as major mediators of TPL-2 transcriptional output in the innate immune response of macrophages, which is particularly important for suppressing the production of type I IFNs. Our earlier experiments have shown that TPL-2 negative regulation of type I IFN signaling is essential for efficient innate immune responses to MTB and L. monocytogenes (21). The present study strongly supports a role for TPL-2 signaling in promoting the innate immune responses to these intracellular bacteria by inducing TCF-dependent Fos transcription to inhibit macrophage production of IFN-b. Type I IFNs also negatively regulate immune responses to Salmonella Typhimurium (69). Furthermore, immune responses to S. Typhimurium are dependent on FOS expression (70). RNA-seq has demonstrated that Map3k8 D270A mutation significantly reduces Fos mRNA upregulation while increasing Ifnb1 mRNA expression after S. Typhimurium infection of BMDMs (L. Blair, F. Breyer, and S.C. Ley, unpublished observations). It will be interesting in future studies to determine whether TPL-2 suppression of type I IFNs is required for efficient innate immune responses to S. Typhimurium. In addition, it will be important to evaluate the role of TPL-2 in innate immune responses to pathogenic bacteria that are dependent on type I IFN signaling, such as Helicobacter pylori and Streptococcus pneumoniae (71).